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Abstract:

Methods for selective extraction and fractionation of algal proteins from
an algal biomass or algal culture are disclosed. A method of selective
removal of products from an algal biomass provides for single and
multistep extraction processes which allow for efficient separation of
algal proteins. These proteins can be used as renewable sources of
proteins for animal feedstocks and human food. Further, lipids remaining
in the algal biomass after extraction of proteins can be used to generate
renewable fuels.

Claims:

1. A method of selectively removing algal proteins from a saltwater algal
biomass or saltwater algal culture, the method comprising: a. harvesting
algae from the saltwater algal biomass or saltwater algal culture; b.
dewatering the harvested algae to remove at least a portion of
extracellular water from the harvested algae, to yield a first biomass
fraction; c. extracting said first biomass fraction with a first solvent,
yielding a second biomass fraction and a first liquid phase; d.
separating said second biomass fraction from said first liquid phase; and
e. concentrating said first liquid phase to yield a mixture enriched in
algal proteins.

9. The method of claim 1, wherein said separation is accomplished by
siphoning or sedimentation.

10. The method of claim 1, wherein said first solvent is added during
said dewatering step.

11. The method of claim 1, further comprising: f. extracting said second
biomass fraction with a second solvent, yielding a third biomass fraction
and a second liquid phase; g. separating said third biomass fraction from
said second liquid phase; and h. concentrating said second liquid phase
to yield a mixture enriched in algal proteins.

12. The method of claim 11, further comprising: i. extracting said third
biomass fraction with a third solvent, yielding a fourth biomass fraction
and a third liquid phase; j. separating said fourth biomass fraction from
said third liquid phase; and k. concentrating said third liquid phase to
yield a mixture enriched in algal proteins.

Description:

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of U.S. patent application Ser.
No. 13/081,213, filed Apr. 6, 2011, entitled Selective Extraction of
Proteins from Saltwater Algae, and claims the benefit of U.S. Provisional
Application No. 61/321,290, filed Apr. 6, 2010, entitled Extraction with
Fractionation of Oil and Proteinaceous Material from Oleaginous Material,
and U.S. Provisional Application No. 61/321,286, filed Apr. 6, 2010,
entitled Extraction With Fractionation of Oil and Co-Products from
Oleaginous Material, the entire contents of which are hereby incorporated
by reference herein.

FIELD OF THE INVENTION

[0002] The invention is concerned with extracting and fractionating algal
products, including, but not limited to, oils and proteins. More
specifically, the systems and methods described herein utilize step
extraction and fractionation with a slightly nonpolar solvent to process
wet algal biomass.

BACKGROUND OF THE INVENTION

[0003] Petroleum is a natural resource composed primarily of hydrocarbons.
Extracting petroleum oil from the earth is expensive, dangerous, and
often at the expense of the environment. Furthermore, world wide
reservoirs of oil are dwindling rapidly. Costs also accumulate due to the
transportation and processing required to convert petroleum oil into
usable fuels such as gasoline and jet fuel.

[0004] Algae have gained a significant importance in recent years given
their ability to produce lipids, which can be used to produce sustainable
biofuel. This ability can be exploited to produce renewable fuels, reduce
global climate change, and treat wastewater. Algae's superiority as a
biofuel feedstock arises from a variety of factors, including high
per-acre productivity compared to typical terrestrial oil crop plants,
non-food based feedstock resources, use of otherwise non-productive,
non-arable land, utilization of a wide variety of water sources (fresh,
brackish, saline, and wastewater), production of both biofuels and
valuable co-products such as carotenoids and chlorophyll.

[0005] Several thousand species of algae have been screened and studied
for lipid production worldwide over the past several decades. Of these,
about 300 species rich in lipid production have been identified. The
lipid composition and content vary at different stages of the life cycle
and are affected by environmental and culture conditions. The strategies
and approaches for extraction are rather different depending on
individual algal species/strains employed because of the considerable
variability in biochemical composition and the physical properties of the
algae cell wall. Conventional physical extraction processes, such as
extrusion, do not work well with algae given the thickness of the cell
wall and the small size (about 2 to about 20 nm) of algal cells.
Furthermore, the large amounts of polar lipids in algal oil, as compared
to the typical oil recovered from seeds, lead to refining issues.

[0006] Upon harvesting, typical algal concentrations in cultures range
from about 0.1-1.0% (w/v). This means that as much as 1000 times the
amount of water per unit weight of algae must be removed before
attempting oil extraction. Currently, existing oil extraction methods for
oleaginous materials strictly require almost completely dry feed to
improve the yield and quality of the oil extracted. Due to the amount of
energy required to heat the algal mass to dry it sufficiently, the algal
feed to biofuel process is rendered uneconomical. Typically, the feed is
extruded or flaked at high temperatures to enhance the extraction. These
steps may not work with the existing equipment due to the single cell
micrometric nature of algae. Furthermore, algal oil is very unstable due
to the presence of double bonded long chain fatty acids. The high
temperatures used in conventional extraction methods cause degradation of
the oil, thereby increasing the costs of such methods.

[0007] It is known in the art to extract oil from dried algal mass by
using hexane as a solvent. This process is energy intensive. The use of
heat to dry and hexane to extract produces product of lower quality as
this type of processing causes lipid and protein degradation.

[0008] Algal oil extraction can be classified into two types: disruptive
or non-disruptive methods.

[0009] Disruptive methods involve cell lies by mechanical, thermal,
enzymatic or chemical methods. Most disruptive methods result in
emulsions, requiring an expensive cleanup process. Algal oils contain a
large percentage of polar lipids and proteins which enhance the
emulsification of the neutral lipids. The emulsification is further
stabilized by the nutrient and salt components left in the solution. The
emulsion is a complex mixture, containing neutral lipids, polar lipids,
proteins, and other algal products, which extensive refining processes to
isolate the neutral lipids, which are the feed that is converted into
biofuel.

[0010] Non-disruptive methods provide low yields. Milking is the use of
solvents or chemicals to extract lipids from a growing algal culture.
While sometimes used to extract algal products, milking may not work with
some species of algae due to solvent toxicity and cell wall disruption.
This complication makes the development of a generic process difficult.
Furthermore, the volumes of solvents required would be astronomical due
to the maximum attainable concentration of the solvent in the medium.

[0011] Multiphase extractions would require extensive distillations, using
complex solvent mixtures, and necessitating mechanisms for solvent
recovery and recycle. This makes such extractions impractical and
uneconomical for use in algal oil technologies.

[0012] Accordingly, to overcome these deficiencies, there is a need in the
art for improved methods and systems for extraction and fractionating
algal products, in particular algal oil, algal proteins, and algal
carotenoids.

BRIEF SUMMARY OF THE INVENTION

[0013] Embodiments described herein relate generally to systems and
methods for extracting lipids of varying polarities from an oleaginous
material, including for example, an algal biomass. In particular,
embodiments described herein concern extracting lipids of varying
polarities from an algal biomass using solvents of varying polarity
and/or a series of membrane filters. In some embodiments, the filter is a
microfilter.

[0014] In some embodiments of the invention, a single solvent and water
are used to extract and fractionate components present in an oleaginous
material. In other embodiments, these components include, but are not
limited to, proteins, polar lipids, and neutral lipids. In still other
embodiments, more than one solvent is used. In still other embodiments, a
mixture of solvents is used.

[0015] In some embodiments, the methods and systems described herein are
useful for extracting coproducts of lipids from oleaginous material.
Examples of such coproducts include, without limitation, proteinaceous
material, chlorophyll, and carotenoids. Embodiments of the present
invention allow for the simultaneous extraction and fractionation of
algal products from algal biomass in a manner that allows for the
production of both fuels and nutritional products.

[0016] Under one embodiment of the invention, a method for selective
extraction of proteins from saltwater algae is provided.

[0017] Under another embodiment of the invention, a method of selectively
extracting algal proteins from a saltwater algal biomass or saltwater
algal culture comprised of substantially intact algal cells includes
heating and mixing the saltwater algal biomass or saltwater algal culture
to generate a first heated extraction mixture comprised of a first
substantially liquid phase, enriched in globulin proteins, and a first
substantially solid phase; separating at least a portion of the first
substantially liquid phase, enriched in globulin proteins, from the first
substantially solid phase; mixing the first solid phase with water and
heating to generate a second heated extraction mixture comprised of a
second substantially liquid phase, enriched in albumin proteins, and a
second substantially solid phase; separating at least a portion of the
second substantially liquid phase, enriched in albumin proteins, from the
second substantially solid phase; mixing the second solid substantially
phase with water and heating to generate a third heated extraction
mixture comprised of a third substantially liquid phase and a third
substantially solid phase; raising the pH of the third heated extraction
mixture to enrich the third substantially liquid phase with glutelin
proteins; separating at least a portion of the third substantially liquid
phase, enriched with glutelin proteins, from the third substantially
solid phase; heating the third substantially solid phase and mixing with
a solvent set to generate a fourth heated extraction mixture comprised of
a fourth substantially liquid phase, enriched in prolamin proteins, and a
fourth substantially solid phase; and separating at least a portion of
the fourth substantially liquid phase, enriched in prolamin proteins,
from the fourth substantially solid phase. In some aspects of the
invention, any one or more of the separating steps is performed by at
least one method selected from the group consisting of centrifugation,
filtration, flotation, and sedimentation. In other aspects of the
invention, at least one of the first, second, third, and fourth heated
extraction mixtures is maintained at a heated temperature for a selected
time prior to separating. In still other aspects of the invention, at
least one of the first, second, third, and fourth heated extraction
mixtures is maintained at a heated temperature for between about 20 to
about 60 minutes. In yet other aspects of the invention, at least one of
the first, second, third, and fourth heated extraction mixtures is
maintained at a heated temperature for between about 45 to about 90
minutes. In some aspects of the invention, at least one of the first,
second, third, and fourth heated extraction mixtures is maintained at
about 50° C. In still other aspects, the fourth substantially
solid phase is enriched in lipids. In other aspects of the invention, the
solvent set comprises ethanol. In still other aspects of the invention,
the solvent set comprises an alcohol.

[0018] In another embodiment of the invention, a method of selectively
extracting globulin proteins from an algal biomass or saltwater algal
culture comprised of substantially intact saltwater algal cells includes
heating and mixing the saltwater algal biomass or saltwater algal culture
to generate a heated liquid phase, enriched in globulin proteins, and a
heated substantially solid phase; and separating at least a portion of
the heated substantially liquid phase, enriched in globulin proteins,
from the heated substantially solid phase. In some aspects of the
invention, the saltwater algal biomass or saltwater algal culture is
maintained at about 50° C. during at least a portion the heating
and mixing.

[0019] In another embodiment of the invention, a method of selectively
extracting albumin proteins from a saltwater algal biomass or saltwater
algal culture comprised of substantially intact algal cells includes
heating and mixing the saltwater algal biomass or saltwater algal culture
to generate a heated substantially liquid phase, enriched in albumin
proteins, and a heated substantially solid phase; separating at least a
portion of the heated substantially liquid phase, enriched in albumin
proteins from the heated substantially solid phase. In some aspects of
the invention, the mixture of saltwater algal biomass or saltwater algal
culture and water is maintained at about 50° C. during at least a
portion of the step of heating and mixing. In some embodiments of the
invention, the solvent set comprises at least one solvent selected from
the group consisting of methanol, ethanol, isopropanol, acetone, ethyl
acetate, and acetonitrile.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020] FIG. 1A is a flowchart of steps involved in a method according to
an exemplary embodiment of the present disclosure.

[0021] FIG. 1B is a schematic diagram of an exemplary embodiment of a
dewatering process according to the present disclosure.

[0022] FIG. 2 is a schematic diagram of an exemplary embodiment of an
extraction system according to the present disclosure.

[0024] FIGS. 4A&B are graphic representations showing neutral lipids (A)
Purity and (B) Recovery in the two step solvent extraction process using
methanol and petroleum ether at three different temperatures.

[0025] FIGS. 5A&B are graphs showing neutral lipids (A) Purity and (B)
Recovery in the two step solvent extraction process using aqueous
methanol and petroleum ether at three different temperatures.

[0026] FIG. 6 is a graph showing lipid recovery in the two step solvent
extraction process using aqueous methanol and petroleum ether at three
different temperatures.

[0027] FIG. 7 is a graph showing the effect of solvents to solid biomass
ratio on lipid recovery.

[0028] FIG. 8 is a graph showing the efficacy of different aqueous
extraction solutions in a single step extraction recovery of aqueous
methanol on dry biomass.

[0029] FIG. 9 is a graph showing the effect of multiple step methanol
extractions on the cumulative total lipid yield and the neutral lipids
purity.

[0030] FIG. 10 is a graph showing the cumulative recovery of lipids using
wet biomass and ethanol.

[0031] FIG. 11 is a graph showing a comparison of the extraction times of
the microwave assisted extraction and conventional extraction systems.

[0032] FIG. 12A is a flowchart of steps involved in a method according to
an exemplary embodiment of the present disclosure which incorporates a
step of protein extraction. All of the units in FIG. 12A are in pounds.

[0033] FIG. 12B is a flowchart of steps involved in an exemplary
extraction process according to the present disclosure.

[0034] FIG. 13 is a flowchart and mass balance diagram describing one of
the embodiments of the present invention wherein 1000 lbs. of algal
biomass was processed through extraction and fractionation in order to
separate neutral lipids, polar lipids, and protein from the algal
biomass.

[0035] FIG. 14 is a flowchart describing one of the embodiments of the
present invention wherein an algal mass can be processed to form various
products.

[0036] FIG. 15 is a flowchart describing one of the embodiments of the
present invention wherein algae neutral lipids are processed to form
various products.

[0037] FIG. 16 is a flowchart describing one of the embodiments of the
present invention wherein algae neutral lipids are processed to form fuel
products.

[0038] FIG. 17 is a flowchart describing one of the embodiments of the
present invention wherein algae proteins are selectively extracted from a
freshwater algal biomass.

[0039] FIG. 18 is a flowchart describing one of the embodiments of the
present invention wherein algae proteins are selectively extracted from a
saltwater algal biomass.

[0040] FIG. 19 is a flowchart describing one of the embodiments of the
present invention wherein a selected algae protein is extracted from a
saltwater or freshwater algal biomass.

[0041] FIG. 20 is a flowchart describing one of the embodiments of the
present invention wherein a selected algae protein is extracted from a
saltwater or freshwater algal biomass.

[0042] FIG. 21 is a photograph showing Scenedescemus sp. cells before and
after extraction using the methods described herein. The cells are
substantially intact both before and after extraction.

DETAILED DESCRIPTION

Definitions

[0043] The term "conduit" or any variation thereof, as used herein,
includes any structure through which a fluid may be conveyed.
Non-limiting examples of conduit include pipes, tubing, channels, or
other enclosed structures.

[0044] The term "reservoir" or any variation thereof, as used herein,
includes any body structure capable of retaining fluid. Non-limiting
examples of reservoirs include ponds, tanks, lakes, tubs, or other
similar structures.

[0045] The term "about" or "approximately," as used herein, are defined as
being close to as understood by one of ordinary skill in the art, and in
one non-limiting embodiment the terms are defined to be within 10%,
preferably within 5%, more preferably within 1%, and most preferably
within 0.5%.

[0046] The terms "inhibiting" or "reducing" or any variation of these
terms, as used herein, includes any measurable decrease or complete
inhibition to achieve a desired result.

[0047] The term "effective," as used herein, means adequate to accomplish
a desired, expected, or intended result.

[0048] The use of the word "a" or "an" when used in conjunction with the
term "comprising" herein may mean "one," but it is also consistent with
the meaning of "one or more," "at least one," and "one or more than one."

[0049] The term "or" as used herein, means "and/or" unless explicitly
indicated to refer to alternatives only or the alternatives are mutually
exclusive, although the disclosure supports a definition that refers to
only alternatives and "and/or."

[0050] The use of the term "wet" as used herein, is used to mean
containing about 50% to about 99.9% water content. Water content may be
located either intracellularly or extracelluarly.

[0051] The use of the term "solvent set" as used herein, is used to mean
composition comprising one or more solvents. These solvents can be
amphipathic (also known as amphiphilic or slightly nonpolar),
hydrophilic, or hydrophobic. In some embodiment, these solvents are water
miscible and in others, they are immiscible in water. Non-limiting
example of solvents that may be used to practice the methods of the
instant invention include methanol, ethanol, isopropanol, acetone, ethyl
acetate, and acetonitrile, alkanes (hexane, pentane, heptane, octane),
esters (ethyl acetate, butyl acetate), ketones (methyl ethyl ketone
(MEK), methyl isobutyl ketone (MIBK)), aromatics (toluene, benzene,
cyclohexane, tetrahydrofuran), haloalkanes (chloroform,
trichloroethylene), ethers (diethyl ether), and mixtures (diesel, jet
fuel, gasoline).

[0052] The term "oil" as used herein includes compositions containing
neutral lipids and polar lipids. The terms "algae oil" and "algal oil" as
used herein are used interchangeably.

[0053] The term "diffusate" or "permeate" as used herein may refer to
material that has passed through a separation device, including, but not
limited to a filter or membrane.

[0054] The term "retentate" as used herein may refer to material that
remains after the diffusate has passed through a separation device.

[0055] As used herein, the words "comprising" (and any form of comprising,
such as "comprise" and "comprises"), "having" (and any form of having,
such as "have" and "has"), "including" (and any form of including, such
as "includes" and "include"), or "containing" (and any form of
containing, such as "contains" and "contain") are inclusive or open-ended
and do not exclude additional, unrecited elements or method steps.

[0056] The term "polar lipids" or any variation thereof, as used herein,
includes, but is not limited to, phospholipids and glycolipids.

[0057] The term "neutral lipids" or any variation thereof, as used herein,
includes, but is not limited to, triglycerides, diglycerides,
monoglycerides, carotenoids, waxes, sterols.

[0058] The term "solid phase" as used herein refers to a collection of
material that is generally more solid than not, and is not intended to
mean that all of the material in the phase is solid. Thus, a phase having
a substantial amount of solids, while retaining some liquids, is
encompassed within the meaning of that term. Meanwhile, the term "liquid
phase", as used herein, refers to a collection of material that is
generally more liquid than not, and such collection may include solid
materials.

[0059] The term "biodiesel" as used herein refers to methyl or ethyl
esters of fatty acids derived from algae

[0060] The term "nutraceutical" as used herein refers to a food product
that provides health and/or medical benefits. Non-limiting examples
include carotenoids, carotenes, xanthophylls such as zeaxanthin,
astaxanthin, and lutein.

[0062] The term "impurities", when used in connection with polar lipids,
as used herein, refers to all components other than the products of
interest that are coextracted or have the same properties as the product
of interest.

[0063] The term "lubricants", when used in connection with polar lipids,
as used herein refers to hydrotreated algal lipids such as C16-C20
alkanes.

[0064] The term "detergents", when used in connection with polar lipids,
as used herein refers to glycolipids, phospholipids and derivatives
thereof.

[0065] The term "food additives", when used in connection with polar
lipids, as used herein refers to soy lecithin substitutes or
phospholipids derived from algae.

[0066] The term "non-glycerin matter" as used herein refers to any
impurity that separates with the glycerin fraction. A further clean up
step will remove most of what is present in order to produce
pharmaceutical grade glycerin.

[0068] Unsaturated long chain fatty acids include, but are not limited to,
omega-3 fatty acids, omega-6 fatty acids, and omega-9 fatty acids. The
term "omega-3 fatty acids" as used herein refers to, but is not limited
to the fatty acids listed in Table 1.

[0069] The term "jet fuel blend stock" as used herein refers to alkanes
with the carbon chain lengths appropriate for use as jet fuels.

[0070] The term "diesel blend stock" as used herein refers to alkanes with
the carbon chain lengths appropriate for use as diesel.

[0071] The term "animal feed" as used herein refers to algae-derived
substances that can be consumed and used to provide nutritional support
for an animal.

[0072] The term "human food" as used herein refers to algae-derived
substances that can be consumed to provide nutritional support for
people. Algae-derived human food products can contain essential
nutrients, such as carbohydrates, fats, proteins, vitamins, or minerals.

[0073] The term "bioremediation" as used herein refers to use of algal
growth to remove pollutants, such as, but not limited to, nitrates,
phosphates, and heavy metals, from industrial wastewater or municipal
wastewater.

[0074] The term "wastewater" as used herein refers to industrial
wastewater or municipal wastewater that contain a variety of contaminants
or pollutants, including, but not limited to nitrates, phosphates, and
heavy metals.

[0075] The term "enriched", as used herein, shall mean about 50% or
greater content.

[0076] The term "substantially", as used herein, shall mean mostly.

[0077] The term "globulin proteins" as used herein refers to salt soluble
proteins.

[0078] The term "albumin proteins" as used herein refers to water soluble
proteins.

[0079] The term "glutelin proteins" as used herein refers to alkali
soluble proteins.

[0080] The term "prolamin proteins" as used herein refers to alcohol
soluble proteins. Non-limiting examples of prolamin proteins are gliadin,
zein, hordein, avenin.

[0081] The term "algal culture" as used herein refers to algal cells in
culture medium.

[0082] The term "algal biomass" as used herein refers to an at least
partially dewatered algal culture.

[0083] The term "dewatered" as used herein refers to the removal of at
least some water.

[0084] The term "algal paste" as used herein refers to a partially
dewatered algal culture having fluid properties that allow it to flow.
Generally an algal paste has a water content of about 90%.

[0085] The term "algal cake" as used herein refers to a partially
dewatered algal culture that lacks the fluid properties of an algal paste
and tends to clump. Generally an algal cake has a water content of about
60% or less.

[0086] Saltwater algal cells include, but are not limited to, marine and
brackish algal species. Saltwater algal cells are found in nature in
bodies of water such as, but not limited to, seas, oceans, and estuaries.
Non-limiting examples of saltwater algal species include Nannochloropsis
sp., Dunaliella sp.

[0087] Freshwater algal cells are found in nature in bodies of water such
as, but not limited to, lakes and ponds. Non-limiting examples of
freshwater algal species include Scendescemus sp., Haemotococcus sp.

[0088] Non-limiting examples of microalgae that can be used with the
methods of the invention are members of one of the following divisions:
Chlorophyta, Cyanophyta (Cyanobacteria), and Heterokontophyta. In certain
embodiments, the microalgae used with the methods of the invention are
members of one of the following classes: Bacillariophyceae,
Eustigmatophyceae, and Chrysophyceae. In certain embodiments, the
microalgae used with the methods of the invention are members of one of
the following genera: Nannochloropsis, Chlorella, Dunaliella,
Scenedesmus, Selenastrum, Oscillatoria, Phormidium, Spirulina, Amphora,
and Ochromonas.

[0090] In other embodiments, the biomass can be plant material, including
but not limited to soy, corn, palm, camelina, jatropha, canola, coconut,
peanut, safflower, cottonseed, linseed, sunflower, rice bran, and olive.

[0091] Systems and methods for extracting lipids and coproducts (e.g.,
proteins) of varying polarity from a wet oleaginous material, including
for example, an algal biomass, are disclosed. In particular, the methods
and systems described herein concern the ability to both extract and
fractionate the algae components by doing sequential extractions with a
hydrophilic solvent/water mixture that becomes progressively less polar
(i.e., water in solvent/water ratio is progressively reduced as one
proceed from one extraction step to the next). In other words, the
interstitial solvent in the algae (75% of its weight) is initially water
and is replaced by the slightly nonpolar solvent gradually to the
azeotrope of the organic solvent. This results in the extraction of
components soluble at the polarity developed at each step, thereby
leading to simultaneous fractionation of the extracted components.
Extraction of proteinaceous byproducts by acid leaching and/or alkaline
extraction is also disclosed.

[0092] In some embodiments of the invention, a single solvent and water
are used to extract and fractionate components present in an oleaginous
material. In other embodiments, a solvent set and water are used to
extract and fractionate components present in an oleaginous material. In
some embodiments the oleaginous material is wet. In other embodiments,
the oleaginous material is algae.

[0093] Polar lipid recovery depends mainly on its ionic charge, water
solubility, and location (intracellular, extracellular or membrane
bound). Examples of polar lipids include, but are not limited to,
phospholipids and glycolipids. Strategies that can be used to separate
and purify polar lipids can roughly be divided into batch or continuous
modes. Examples of batch modes include precipitation (pH, organic
solvent), solvent extraction and crystallization. Examples of continuous
modes include centrifuging, adsorption, foam separation and
precipitation, and membrane technologies (tangential flow filtration,
diafiltration and precipitation, ultra filtration).

[0094] Other objects, features and advantages of the present invention
will become apparent from the following detailed description. It should
be understood, however, that the detailed description and the examples,
while indicating specific embodiments of the invention, are given by way
of illustration only. Additionally, it is contemplated that changes and
modifications within the spirit and scope of the invention will become
apparent to those skilled in the art from this detailed description.

[0095] Surprisingly, the proposed non-disruptive extraction process
results in over 90% recovery. The small amount of polar lipids in the
remaining biomass enhances its value when the remaining biomass is used
for feed. This is due, at least in part, to the high long chain
unsaturated fatty acid content of the biomass. In addition, ethanol
extracts can further be directly transesterified. Furthermore, unlike the
existing conventional methods, the methods and systems described herein
are generic for any algae, and enable recovery of a significant portion
of the valuable components, including polar lipids, in the algae by the
use of a water miscible organic solvent gradient.

[0096] The neutral lipid fraction obtained by the use of the present
invention possesses a low metal content, thereby enhancing stability of
the lipid fraction, and reducing subsequent processing steps. Metals tend
to make neutral lipids unstable due to their ability to catalyze
oxidation. Furthermore, metals inhibit hydrotreating catalysts,
necessitating their removal before a neutral lipid mixture can be
refined. The systems and methods disclosed herein allow for the
extraction of metals in the protein and/or the polar lipid fractions.
This is advantageous because proteins and polar lipids are not highly
affected by metal exposure, and in some cases are actually stabilized by
metals.

[0097] The systems and methods disclosed herein can start with wet
biomass, reducing the drying and dewatering costs. Compared to
conventional extraction processes, the disclosed extraction and
fractionation processes should have relatively low operating costs due to
the moderate temperature and pressure conditions, along with the solvent
recycle. Furthermore, conventional extraction processes are cost
prohibitive and cannot meet the demand of the market.

[0098] Another aspect of the systems and methods described herein is the
ability to accomplish preliminary refining, which is the separation of
polar lipids from neutral lipids during the extraction process. The
differences between algal oil used in exemplary embodiments and vegetable
oils used in previous embodiments include the percentage of individual
classes of lipids. An exemplary algal crude oil composition is compared
with vegetable oil shown in Table 2 below:

[0099] Degumming (physical and/or chemical) of vegetable oil is done in
order to remove polar lipids (e.g., glycolipids and phospholipids).
Vegetable oil that has been chemically degummed retains a significant
quantity of neutral lipid. This neutral lipid fraction is further removed
from the degummed material using solvent extraction or
supercritical/subcritical fluid extraction or membrane technology. In
contrast, separation of the neutral lipids from an oleaginous algal
biomass is far more difficult than from a vegetable oil feedstock due to
the presence of large quantities of polar lipids typically found in algal
oil (see Table 2). This is because the larger percentage of polar lipids
present in algal oil enhances the emulsification of the neutral lipids.
The emulsification is further stabilized by the nutrient and salt
components left in the solution. The presence of polar lipids, along with
metals, results in processing difficulties for separation and utilization
of neutral lipids. However, because polar lipids have an existing market,
their recovery would add significant value to the use of algal oil to
generate fuels.

[0100] Polar lipids are surfactants by nature due to their molecular
structure and have a huge existing market. Many of the existing
technologies for producing polar lipids are raw material or cost
prohibitive. Alternative feedstocks for glycolipids and phospholipids are
mainly algae oil, oat oil, wheat germ oil and vegetable oil. Algae oil
typically contains about 30-85% (w/w) polar lipids depending on the
species, physiological status of the cell, culture conditions, time of
harvest, and the solvent utilized for extraction. Further, the glycerol
backbone of each polar lipid has two fatty acid groups attached instead
of three in the neutral lipid triacylglycerol. Transesterification of
polar lipids may yield only two-thirds of the end product, i.e.,
esterified fatty acids, as compared to that of neutral lipids, on a per
mass basis. Hence, removal and recovery of the polar lipids would not
only be highly beneficial in producing high quality biofuels or
triglycerides from algae, but also generate value-added co-products
glycolipids and phospholipids, which in turn can offset the cost
associated with algae-based biofuel production. The ability to easily
recover and fractionate the various oil and co-products produced by algae
is advantageous to the economic success of the algae oil process.

[0101] A further aspect of the methods and systems described herein is the
ability to extract proteins from an oleaginous material, such as algal
biomass. The methods disclosed herein of extraction of proteinaceous
material from algal biomass comprise a flexible and highly customizable
process of extraction and fractionation. For example, in some
embodiments, extraction and fractionation occur in a single step, thereby
providing a highly efficient process. Proteins sourced from such biomass
are useful for animal feeds, food ingredients and industrial products.
For example, such proteins are useful in applications such as fibers,
adhesives, coatings, ceramics, inks, cosmetics, textiles, chewing gum,
and biodegradable plastics.

[0102] Another aspect of the methods and systems described herein involves
varying the ratio of algal biomass to solvent based on the components to
be extracted. In one embodiment, an algal biomass is mixed with an equal
weight of solvent. In another embodiment, an algal biomass is mixed with
a lesser weight of solvent. In yet another embodiment, an algal biomass
is mixed with a greater weight of solvent. In some embodiments, the
amount of solvent mixed with an algal biomass is calculated based on the
solvent to be used and the desired polarity of the algal biomass/solvent
mixture. In still other embodiments, the algal mass is extracted in
several steps. In an exemplary embodiment, an algal biomass is
sequentially extracted, first with about 50-60% of its weight with a
slightly nonpolar, water miscible solvent. Second, the remaining algal
solids are extracted using about 70% of the solids' weight in solvent. A
third extraction is then performed using about 90% of the solid's weight
in solvent. Having been informed of these aspects of the invention, one
of skill in the art would be able to use different solvents of different
polarities by adjusting the ratios of algal biomass and/or solid
residuals to the desired polarity in order to selectively extract algal
products.

[0103] For example, in preferred embodiment, the solvent used is ethanol.
Components may be selectively isolated by varying the ratio of solvent.
Proteins can be extracted from an algal biomass with about 50% ethanol,
polar lipids with about 80% ethanol, and neutral lipids with about 95% or
greater ethanol. If methanol were to be used, the solvent concentration
to extract proteins from an algal biomass would be about 70%. Polar
lipids would require about 90% methanol, and neutral lipids would require
about 100% methanol.

[0104] Embodiments of the systems and methods described herein exhibit
surprising and unexpected results. First of all, the recovery/extraction
process can be done on a wet biomass. This is a major economic advantage
as exemplary embodiments avoid the use of large amounts of energy
required to dry and disrupt the cells. Extraction of neutral lipids from
a dry algal biomass is far more effective using the systems and methods
of the present invention. The yields obtained from the disclosed
processes are significantly higher and purer than those obtained by
conventional extractions. This is because conventional extraction
frequently results in emulsions, rendering component separations
extremely difficult.

[0105] Exemplary embodiments may be applied to any algae or non-algae
oleaginous material. Exemplary embodiments may use any water-miscible
slightly nonpolar solvent, including, but not limited to, methanol,
ethanol, isopropanol, acetone, ethyl acetate, and acetonitrile. Specific
embodiments may use a green renewable solvent, such as ethanol. The
alcohol solvents tested resulted in higher yield and purity of isolated
neutral lipids. Ethanol is relatively economical to purchase as compared
to other solvents disclosed herein. In some exemplary embodiments,
extraction and fractionation can be performed in one step followed by
membrane-based purification if needed. The resulting biomass is almost
devoid of water and can be completely dried with lesser energy than an
aqueous algae slurry.

[0106] In some exemplary embodiments, the solvent used to extract is
ethanol. Other embodiments include, but are not limited to, cyclohexane,
petroleum ether, pentane, hexane, heptane, diethyl ether, toluene, ethyl
acetate, chloroform, dicholoromethane, acetone, acetonitrile,
isopropanol, and methanol. In some embodiments, the same solvent is used
in sequential extraction steps. In other embodiments, different solvents
are used in each extraction step. In still other embodiments, two or more
solvents are mixed and used in one or more extraction steps.

[0107] In some embodiments of the methods described herein, a mixture of
two or more solvents used in any of the extraction steps includes at
least one hydrophilic solvent and at least one hydrophobic solvent. When
using such a mixture, the hydrophilic solvent extracts the material from
the biomass via diffusion. Meanwhile, a relatively small amount of
hydrophobic solvent is used in combination and is involved in a
liquid-liquid separation such that the material of interest is
concentrated in the small amount of hydrophobic solvent. The two
different solvents then form a two-layer system, which can be separated
using techniques known in the art. In such an implementation, the
hydrophobic solvent can be any one or more of an alkane, an ester, a
ketone, an aromatic, a haloalkane, an ether, or a commercial mixture
(e.g., diesel, jet fuel, gasoline).

[0108] In some embodiments, the extraction processes described herein
incorporate pH excursion in one or more steps. Such pH excursion is
useful for isolating proteinaceous material. In some embodiments, the pH
of the extraction process is acid (e.g., less than about 5). In some
embodiments, the pH of the extraction process is alkaline (e.g., greater
than about 10).

[0109] The use of hexane in conventional extraction procedures
contaminates algal biomass such that coproducts may not be used in food
products. Embodiments of the present invention are superior to those
known in the art as they require the use of far less energy and render
products suitable for use as fuels as well as foodstuffs and nutrient
supplements.

[0110] It is contemplated that any embodiment discussed in this
specification can be implemented with respect to any method or system of
the invention, and vice versa. Furthermore, systems of the invention can
be used to achieve methods of the invention.

DESCRIPTION OF EXEMPLARY EMBODIMENTS

[0111] For solvent extraction of oil from algae the best case scenario is
a solvent which selectively extracts triacylglycerols (TAG) and leaving
all polar lipids and non-TAG neutral lipids such as waxes, sterols in the
algal cell with high recoveries. The second option would be selectively
extract polar lipids and then extract purer neutral lipids devoid of
polar lipids, resulting in high recovery. The last option would be to
extract all the lipids and achieve very high recovery in one or two
steps.

[0112] Referring now to FIG. 1A, a flowchart 100 provides an overview of
the steps involved in exemplary embodiments of methods used in the
fractionation and purification of lipids from an algae-containing
biomass. In a first step 110, algal cells are harvested. In a subsequent
step 120, water is removed from algal cells to yield a 10-25% solid
biomass. In step 130, a solvent-based extraction is performed on the
biomass and the fractions are collected. In some embodiments, step 130
will also incorporate pH-based extraction and fraction collection.
Finally, a solid/liquid phase separation, including, but not limited to
techniques such as filtration, decanting, and centrifugation, may be
performed in a step 140 to in order to separate out smaller lipid
components.

[0113] The algae biomass when harvested in step 110 typically consists of
about 1-5 g/L of total solids. The biomass can be partially dewatered in
step 120 using techniques including, but not limited to, dissolved air
floatation, membrane filtration, flocculation, sedimentation, filter
pressing, decantation or centrifugation. Dewatering is the removal of
some, most, or all of the water from a solid or semisolid substance.
Embodiments of the present invention utilize dewatering techniques to
remove water from a harvested algal biomass. Dewatering can be carried
out using any one of or a combination of any of the methods described
herein, as well as by any other methods known to one of skill in the art.

[0114] The dewatered algae biomass resulting from step 120 typically
consists of about 10-30% solids. This biomass can then be extracted with
water miscible slightly nonpolar solvents (e.g., alcohols), in a
multistage countercurrent solvent extraction process segregating the
fractions at each stage. This type of process can reduce both capital and
operating expenses. In some embodiments, the biomass also undergoes acid
and/or alkaline extraction to fractionate protein material.

[0115] In some embodiments, dewatering of an algal biomass can be carried
out by treating the harvested algal biomass with a solvent such as
ethanol. The algal biomass is then allowed to settle out of solution and
the liquids may then be removed by methods such as, but not limited to,
siphoning. This novel method of dewatering has lower capital and
operating costs than known methods, enables solvent recycling, reduces
the cost of drying the biomass, and has the added benefit of decreasing
the polarity of the algal biomass prior to beginning extraction and/or
separation of algal components. In fact, it is theorized that the
solvent-based sedimentation processes described herein are effective, in
part, due to the fact that organic solvents reduce or neutralize the
negative charge on the algae surface. In some embodiments of the
invention, dewatering methods are combined in order to remove even more
water. In some embodiments, the addition of solvent during the dewatering
process begins the process of extraction.

[0116] FIG. 1B shows an illustrative implementation of a dewatering
process 300. An algal culture 310 having a final dry weight of about 1
g/L to about 10 g/L (i.e., 0.1-1% w/w) is subjected to a water separation
process 320. Process 320 can include centrifugation, decanting, settling,
or filtration. In one embodiment, a sintered metal tube filter is used to
separate the algal biomass from the water of the culture. When using such
a filter, the recovered water 330 is recycled directed to other algae
cultures. Meanwhile, the algal biomass recovered has been concentrated to
an "algae paste" with a algae density as high as about 200 g/L (i.e.,
10-20% w/w). This concentrated algae paste is then treated with a solvent
340 in a solvent-based sedimentation process 350.

[0117] Sedimentation process 350 involves adding solvent 340 to the algae
paste to achieve a mixture having a weight/weight solvent to biomass
ratio of between about 1:1 to about 1:10. The algae is allowed to settle
in a settling vessel, and a solvent/water mixture 360 is removed by, for
example, siphoning and/or decanting. The solvent can be recovered and
reused by well-known techniques, such as distillation and/or
pervaporation. The remaining wet biomass 370 is expected to have a solids
content of about 30% to about 60% w/w in an alcohol and water solution.

[0118] Solvents ideal for dewatering are industrially common water-soluble
solvents with densities over 1.1 g/mL or below 0.9 g/mL. Examples include
isopropanol, acetone, acetonitrile, t-butyl alcohol, ethanol, methanol,
1-propanol, heavy water (D2O), ethylene glycol, and/or glycerin. If
the solvent density is over 1.1 g/mL then the algae biomass would float
rather than create a sediment at the bottom of the settling vessel.

[0119] FIG. 2 is a schematic diagram of an exemplary embodiment of an
extraction system 200. The wet or dry algal biomass is transported using
methods known in the art, including, but not limited to a moving belt, a
screw conveyor, or through extraction chambers. The solvent for
extraction is recirculated from a storage tank assigned to each biomass
slot position. The extraction mixture is filtered, returning the biomass
solids back into the slot and the extract into the storage tank. The
solids on the belt move periodically based on the residence time
requirement for extraction. The extracts in each storage tank may either
be replenished at saturation or continuously replaced by fresh solvent.
This would also reduce the downstream processing time and cost
drastically. This embodiment comprises a primary reservoir 210, a
transport mechanism 220, a plurality of separation devices 241-248 (e.g.,
membrane filtration devices), a plurality of extraction reservoirs
261-268, and a plurality of recycle pumps 281-287. In this embodiment,
primary reservoir 210 is divided up into a plurality of inlet reservoirs
211-218.

[0120] During operation, algal biomass 201 is placed a first inlet
reservoir 211 near a first end 221 of transport mechanism 220. In
addition, solvent 205 is placed into inlet reservoir 218 near a second
end 222 of transport mechanism 220. Transport mechanism 220 directs the
algal biomass along transport mechanism 220 from first end 221 towards
second end 222. As the algal biomass is transported, it passes through
the plurality of separation devices 241-248 and is separated into
fractions of varying polarity. The diffusate portions that pass through
separation devices 241-248 are directed to reservoirs 261-268.

[0121] For example, the diffusate portion of the algal biomass that passes
through the first separation device 241 (e.g., the portion containing
liquid and particles small enough to pass through separation device 241)
is directed to the first reservoir 261. From first reservoir 261, the
diffusate portion can be recycled back to first inlet reservoir 201. The
retentate portion of the algal biomass that does not pass through first
separation device 241 can then be directed by transport mechanism 220 to
second inlet reservoir 212 and second separation device 242, which can
comprise a finer separation or filtration media than the first separation
device 241.

[0122] The segment of the diffusate portion that passes through second
separation device 242 can be directed to second reservoir 262, and then
recycled back to second inlet reservoir 212 via recycle pump 282. The
retentate or extracted portion of the algal biomass that does not pass
through second separation device 242 can be directed by transport
mechanism 220 to third inlet reservoir 213. This process can be repeated
for inlet reservoirs 213-218 and separation devices 243-248 such that the
retentate portions at each stage are directed to the subsequent inlet
reservoirs, while the diffusate portions are directed to the recycle
reservoirs and recycled back to the current inlet reservoir.

[0123] In exemplary embodiments, the first fraction will be extracted with
the highest water to slightly nonpolar solvent ratio, i.e., most polar
mixture, while the last fraction will be extracted with the most pure
slightly nonpolar solvent, i.e. the least polar mixture. The process
therefore extracts components in the order of decreasing polarity with
the fraction. The function of the first fraction is to remove the
residual water and facilitate the solvent extraction process. The
fractions that follow are rich in polar lipids, while the final fractions
are rich in neutral lipids.

[0124] The oil fraction can be esterified to liberate the long chain
unsaturated fatty acids. The carotenoids and long chain unsaturated fatty
acids can be separated from the oil using processes such as molecular
distillation in conjunction with non-molecular distillation. All of the
fatty acids can be separated from the carotenoids using the molecular
distillation. The distillates can be fractionated using a simple
distillation column to separate the lower chain fatty acids for refining.
The long chain unsaturated fatty acids remain as high boiling residue in
the column.

[0125] In some non-limiting embodiments, the extraction system and methods
described herein incorporate one or more steps to isolate protein
material from the oleaginous material (e.g., algal biomass). Such protein
extraction steps employ pH adjustment(s) to achieve isolation and
extraction of protein. For example, in one non-limiting embodiment, the
pH of the solvent in the first separation device is optimized for protein
extraction, resulting in a first fraction that is rich in protein
material. The pH of the protein extraction step is adjusted depending on
the pKa of the proteins of interest. The pKa of a protein of interest may
be ascertained using methods known to one of skill in the art, including,
but not limited to using the Poisson-Boltzmann equation, empirical
methods, molecular dynamics based methods, or the use of titration
curves.

[0126] In some embodiments, the solvent pH is alkaline. For example, in
some embodiments, the solvent pH is greater than about 10. In other
embodiments, the solvent pH ranges from about 10 to about 12. In further
embodiments, the solvent pH is about 10, about 11, or about 12. In other
embodiments, the solvent pH is acid. For example, in some embodiments,
the solvent pH is less than about 5. In other embodiments, the solvent pH
ranges from about 2 to about 5. In further embodiments, the solvent pH is
about 2, about 3, about 4, about 4.5, or about 5. The extracted portion
of the first separation device is then directed to subsequent inlet
reservoirs to achieve extraction and fractionation based on polarity. In
another non-limiting embodiment, protein material is separated in the
final separation device by similar means (i.e., solvent pH adjustment).

[0127] Adjustment of solvent pH is accomplished in accordance with methods
known to those of skill in the art. For example, acid pH is achieved by
mixture of an appropriate acid into the solvent stream. Exemplary acids
useful for protein extraction include, without limitation, phosphoric
acid, sulfuric acid, and hydrochloric acid. Similarly, alkaline pH is
achieved by addition and mixture of an appropriate base into the solvent
stream. Exemplary bases useful for protein extraction include, without
limitation, potassium hydroxide, and sodium hydroxide.

[0128] In some embodiments, protein extraction is performed in a system
separate from the extraction and fractionation system described herein.
For example, in some embodiments, an algal biomass is soaked in a
pH-adjusted solvent mixture, followed by isolation via an appropriate
separation technique (e.g., centrifugation, or filtration). The remaining
solid is then introduced into an extraction and fractionation system
based on polarity, as described herein. Similarly, in some embodiments,
the remaining extract from an extraction and fractionation process based
on polarity is exposed to a pH-adjusted solvent mixture to isolate
protein material at the end of the extraction process.

[0129] As shown in FIG. 3, the solvent selection and the theory of
fractionation based on polarity were developed by extensive analysis of
solvents and the effect on extraction using the Sohxlet extraction
process, which allows the separation of lipids from a solid material. The
Sohxlet extraction system was utilized for rapid screening solvents for
lipid class selectivity and recovery. Solvents from various chemical
classes encompassing a wide range of polarities such as alkanes,
cycloalkane, alkyl halides, esters, ketones, were tested. Prior to the
extraction, the lipid content and composition of the biomass to be
extracted was tested in triplicate using the standard methods for algae
oil estimation such as the Bligh-Dyer lipid extraction method. The
biomass contained 22.16% total lipid, of which 49.52% was neutral lipid.

[0130] FIG. 3 presents the data gathered by extraction of a dry algal mass
using various polar and nonpolar solvents combined with a Sohxlet
extraction process. Depending on the chain length of the alkane solvent,
60-70% purity of neutral lipids and 15-45% of total lipid recovery can be
achieved without disruption and solvent extraction. The longest chain
alkane solvent tested, heptane, recovered 60% of the neutral lipids and
42% of the total lipid. As FIG. 3 shows, the results of extraction of dry
algal mass using solvents and conventional extraction methods such as
hexane are inefficient, expensive, and result in poor yields. The systems
and methods discloses herein address these inefficiencies by controlling
the proportion of slightly nonpolar solvent to water in order to separate
out components of differing polarities with minimal loss of components.

[0131] The lower carbon alcohol solvents were more selective for polar
lipids. The neutral lipid purity was 22% for methanol and 45% for
ethanol. Isopropyl alcohol did not show any selectivity between polar and
nonpolar lipids, resulting in a 52% pure neutral lipid product. Methanol
recovered 67% of the total lipids and more than 90% of the polar lipids.
Therefore, methanol is an excellent candidate for an embodiment of the
present invention wherein methanol can be used to selectively extract
polar lipids from an oleaginous material prior to extracting the neutral
lipids using heptane or hexane. The other solvent classes tested did not
show any selectivity towards lipid class since the neutral lipid purity
was close to 49%, similar to the lipid composition present in the
original biomass. Furthermore, the total lipid recovery achieved with
these solvents ranged from about 15-35%, rendering these solvents
unsuitable for the selective extraction of particular lipid classes or
total lipid extraction.

[0132] The results from the Sohxlet analysis were confirmed using the
standard bench scale batch solvent extraction apparatus described below
in Example 1. The solvents selected were methanol for the first step to
recover polar lipids, and petroleum ether in the second step for recovery
of neutral lipids. All of the extractions were performed with a 1:10
solid:solvent ratio. Each extraction step in this experiment was 1 hour
long. Other experiments done (data not shown) indicate that about 45
minutes or longer is long enough for the extraction to be successful.
This retention time is dependent on the heat and mass transfer of the
system.

[0133] The methanol extractions were performed at different temperatures,
40° C., 50° C., and 65° C., in order to determine
which was optimal. The petroleum ether extraction was performed at
35° C., close to the boiling point of the solvent. Petroleum ether
was chosen because of its high selectivity for neutral lipids, low
boiling point, and the product quality observed after extraction.

[0134] FIG. 4A shows that the neutral lipid purity in a petroleum ether
extraction carried out after a methanol extraction step at 65° C.
is over 80%, demonstrating that the combination of these two extraction
steps enhanced the neutral lipid content of the final crude oil product.
FIG. 4B shows that the total neutral lipid recovery was low and there was
a significant amount of neutral lipid loss in the first step.

[0135] To minimize the loss of neutral lipids in the methanol extraction
step, the polarity of the solvent can be increased by adding water to the
solvent. FIGS. 5A and 5B show the results of extracting the
aforementioned biomass with 70% v/v aqueous methanol followed by
extraction with petroleum ether. FIG. 5A shows that the neutral lipid
purity was much higher in the petroleum ether extraction than was
achieved by the use of pure methanol. Moreover, the loss of neutral
lipids was greatly reduced by the use of aqueous methanol in the first
extraction step. As seen in FIG. 5B, methanol extraction at higher
temperatures improved neutral lipid purity but slightly decreased the
total lipid recovery in the subsequent step.

[0136] In some exemplary embodiments the temperature of the extraction
process is controlled in order to ensure optimal stability of algal
components present in the algal biomass. Algal proteins, carotenoids, and
chlorophyll are examples of algal components that exhibit temperature
sensitivity. In other embodiments, the temperature is increased after the
temperature sensitive algal components have been extracted from the algal
biomass.

[0137] In still other exemplary embodiments, the temperature of the
extraction process is adjusted in order to optimize the yield of the
desired product. Extractions can be run from ambient temperature up to,
but below, the boiling point of the extraction mixture. In still other
embodiments, the temperature of the extraction process is changed
depending on the solubility of the desired product. In still other
embodiments, the extraction temperature is optimized depending on the
algal strain of the biomass to be extracted. Elevated extraction
temperatures increase the solubility of desired compounds and reduce the
viscosity of the extraction mixture enhancing extraction recovery.

[0138] In some embodiments, the extraction is run under pressure to
elevate the boiling point of the extraction mixture. In these
implementations, the pressure is increased to the degree necessary to
prevent boiling, while maintaining the temperature of the extraction
mixture below a temperature at which any of the desired products would
begin to degrade, denature, decompose, or be destroyed.

[0139] In some exemplary embodiments, the extraction is performed near the
boiling point of the solvent used, at the conditions under which the
extraction is performed (e.g., atmospheric or elevated pressures). In
other embodiments, the extraction is performed near the boiling point of
the extraction mixture, again accounting for other extraction conditions.
At such temperatures, vapor phase penetration of the solvent into the
algal cells is faster due to lower mass transfer resistance. If the
extraction temperature is allowed to significantly exceed the boiling
point of the solvent, the solvent-water system can form an azeotrope.
Thus, maintaining the system at or near the boiling point of solvent
would generate enough vapors to enhance the extraction, while reducing
expense. In addition, the solubility of oil is increased at higher
temperatures, which can further increase the effectiveness of extraction
at temperatures close to the solvent boiling point. FIG. 6 shows the
total lipid recovery in the aqueous methanol-petroleum ether extraction
scheme. Although performing the methanol extraction near its boiling
temperature slightly decreases the neutral lipid recovery as observed in
FIG. 5B, it enhances the total lipid recovery.

[0140] In other embodiments, the extraction is carried out under ambient
lighting conditions. In other embodiments, the extraction is carried out
in an opaque container such as, but not limited to, a steel tube or
casing, in order to protect light sensitive algal components from
degradation. Carotenoids are light sensitive algal components.

[0141] In other exemplary embodiments, the extraction takes place under
normal atmospheric conditions. In still other embodiments, the extraction
takes place under a nitrogen atmosphere in order to protect algal
components prone to oxidation. In still other embodiments, the extraction
takes place under an atmosphere of inert gas in order to protect algal
components prone to oxidation. Algal components that might be prone to
oxidation include carotenoids, chlorophyll, and lipids.

[0142] In exemplary embodiments, the solvent-to-solid ratio for the
extraction is between 3-5 based on the dry weight of the solids in the
biomass. The residual algal biomass is rich in carbohydrates (e.g.,
starch) and can be used as a feed stock to produce the solvent used for
extraction.

[0143] FIG. 7 shows the effect of the solvent to solid ratio on the total
lipid recovery. As the solvent to solid ratio was increased, there was a
corresponding and drastic increase in total lipid recovery. It is
believed that this was because of the lower solubility of lipids in
methanol as compared to other commonly used oil extraction solvents such
as hexane.

[0144] The solubility of components is affected by the polarity of solvent
used in an extraction process. The solubility properties can be used to
determine the ratio of wet biomass to solvent. For example, a 40% w/w wet
biomass has 40 g biomass and 60 g water for every 100 g of wet biomass.
If 100 g of ethanol is added to this mixture, the ratio of ethanol to wet
biomass is 1 part wet biomass to 1 part ethanol and the concentration of
ethanol in the mixture is 100/(100+60) equals about 62% w/w of ethanol in
the liquid phase. 62% w/w of ethanol in ethanol water mixture corresponds
to a polarity index of 6.6, calculated by weight and averaging the
polarities of the components. Ethanol, having a polarity index of 5.2,
and water, having a polarity index of 9, in a mixture containing 62%
ethanol and 38% water results in a polarity index of (0.62*5.2+0.38*9)
about 6.6. The polarity index of the mixture for extraction of polar
lipids and neutral lipids is calculated to be about 5.8 and 5.4
respectively. In light of the instant disclosure, one of skill in the art
would be able to formulate a solvent set that can selectively extract
these components.

[0145] In another example, if the extraction solvent is a 1:1 mixture of
isopropyl alcohol and ethanol, the polarity of this solvent is
((3.9+5.4)/2) which is about 4.65. The ratio of solvent to wet biomass
would be calculated to match the polarities. To get a 6.6 polarity index,
we would need to make a 55% w/w of IPA-water mixture calculated by
solving the following algebraic equation:

##STR00001##

[0146] With a 40% w/w wet biomass this would correspond to a ratio of 100
parts wet biomass to 75 parts solvent mixture. A 40% w/w wet biomass has
40 g biomass and 60 g water for every 100 g of wet biomass. If 75 g of
solvent mixture is added to this mixture, the concentration of solvent in
the mixture is (75/(75+60)) is about 55% w/w of solvent mixture in the
solvent mixture-water solution. This calculation can be used to obtain
the solvent biomass ratio at each extraction stage and for each product.
A few nonlimiting examples of solvent sets appear in Table 3.

[0147] The extraction mixture described in all examples, is made up of a
substantially solid phase and a substantially liquid phase. These phases
are then separated post extraction. This can then be followed by removal
of the liquid solvent from the liquid phase, yielding an extraction
product. In some embodiments, the solvent is evaporated. In such an
implementation, a liquid-liquid extraction technique can be used to
reduce the amount of solvent that needs to be evaporated. Any solvents
used can be recycled if conditions allow.

[0148] It was theorized that treatment of the algal biomass prior to
extraction would enhance the productivity and efficiency of lipid
extraction. In this direction an experiment was done comparing the effect
of adding a base or another organic solvent to an algal biomass to change
the surface properties and enhance extraction. A variety of treatments
including aqueous methanol, aqueous sodium hydroxide, and aqueous DMSO
were attempted. As FIG. 8 demonstrates, the addition of 5% DMSO increases
the lipid recovery 3-fold. These extraction steps may be exploited to
dramatically reduce the methanol extraction steps. However, the solutions
used in the above experiments may not be ideal for use on larger scales
due to the high cost, viscosity, and ability to recover and recycle DMSO.

[0149] FIG. 9 is a chart showing the effect of an eight step methanol
extraction on the cumulative total lipid yield and the purity of the
extracted neutral lipid. In this embodiment, 112 grams of wet biomass
(25.6% dry weight), was extracted with 350 mL pure methanol and heating
for 10 minutes at 160 W irradiance power in each step. This resulted in
an extraction temperature of about 75° C., which was near the
boiling point of the extraction mixture. Using this process, it was
determined that it is possible to obtain highly pure neutral lipids from
algal oil once the majority of the polar lipids have been extracted. FIG.
9 shows that it is possible to isolate high purity neutral lipid once the
polar lipids are all extracted. In this case a 5% yield of total biomass
was achieved with over 90% neutral lipids purity in methanol extraction
steps 5 through 8. Furthermore, due to the boiling point of the
extraction mixture, most of the water in the biomass is completely
extracted in the first extraction step, along with carbohydrates,
proteins and metals.

[0150] FIG. 10 shows that recovery of lipids can be made more efficient by
the use of ethanol to extract lipids and protein from wet biomass. By
using ethanol, 80% total lipid recovery can be achieved in about 4 steps
rather than the 9 generally needed by using methanol. This increase in
recovery may be attributed to greater solubility of lipids in ethanol as
compared to methanol. Furthermore, the boiling point of aqueous ethanol
is higher than aqueous methanol, facilitating further recovery of lipids.
This is because the higher temperature renders the oil less viscous,
thereby improving diffusability. Another distinct advantage of this
process is using the residual ethanol in the oil fraction for
transesterification as well as lowering the heat load on the biomass
drying operation.

[0151] Further, FIG. 10 demonstrates that the initial fractions are
non-lipid rich, containing proteins and other highly polar molecules,
followed by the polar lipid rich fractions and finally the neutral lipid
fractions. Hence with a proper design of the extraction apparatus, one
can recover all the three products in a single extraction and
fractionation process.

[0152] Another embodiment of the current invention utilizes microwaves to
assist extraction. Based on previously gathered data disclosed in this
application, it is shown that methanol is the best single solvent for
extraction of all lipids from algae. Hence, a single solvent multiple
step extraction, as described in Example 1 of the instant application,
was performed in order to gather data on the efficacy of a one solvent
microwave extraction system.

[0153] FIG. 11 is a logarithmic plot comparing the extraction time and
total lipid recovery of conventional extraction and microwave-assisted
extraction. Based on the slope of the curve, it was calculated that the
microwave system reduces the extraction time by about five fold or more.
While the conventional methods have a higher net lipid recovery, this is
due to higher recoveries of polar lipids. Based on these results, the
conditions for extraction of dry algal biomass using solvents with and
without microwave assistance have been optimized. Some embodiments of the
invention use traditional microwave apparatus, which emit wavelengths
that excite water molecules. Further embodiments of the invention utilize
customized microwave apparatus capable of exciting different solvents.
Still other embodiments of the invention utilize custom microwave
apparatus capable of exciting the lipids present in the algal biomass. In
some embodiments, the lipids present in the algal biomass are excited
using microwaves, thereby enhancing the separation and extraction of the
lipid components from the algal biomass.

[0154] Moisture content is another parameter of biomass that will
influence the efficiency of oil extraction. In some embodiments of the
present invention, dry algal mass is extracted and fractionated. In other
embodiments, the algal mass is wet. Biomass samples with algae mass
contents of 10%, 25%, and 33% were used to investigate the influence of
moisture on extraction performance.

[0155] FIG. 12A shows an illustrative process 400 for a step-wise
extraction of products from an algae biomass. All units in FIG. 12A are
in pounds. FIG. 12A shows a mass balance of the process 400, while the
details of the equipment and/or systems for performing the process are
described elsewhere herein. A biomass containing 5 pounds of algae has
about 0.63 pounds of polar lipids, 1.87 pounds neutral lipids, 1 pound
protein, and 1.5 pounds carbohydrates. The biomass and 1000 pounds of
water is processed in a dewatering step 405, which separates 950 pounds
of water from the mixture and passes 5 pounds of algae in 45 pounds of
water to a first extraction step 410. Any of the dewatering techniques
disclosed herein can be used tin dewatering step 405. In the first
extraction step 410, 238 pounds of ethanol and 12 pounds of water are
combined with the algae and water from the previous step. The first
extraction step 410 has a liquid phase of about 80.9% w/w ethanol. A
first liquid phase of 231 pounds of ethanol, 53 pounds of water, and 0.5
pounds of algal proteins are recovered, from which water and ethanol are
removed by, e.g., evaporation, leaving a protein-rich product 415.
Solvent recovered from the evaporation can be recycled to the first
extraction step 410.

[0156] A first solid phase from the first extraction step 410 is passed to
a second extraction step 420; this first solid phase includes 4.5 pounds
of algae, 2.6 pounds of water, and 10.9 pounds of ethanol. Eighty-six
pounds of ethanol and 4 pounds of water are added to the first solid
phase from the previous step. The second extraction step 420 has a liquid
phase of about 93.6% w/w ethanol. A second liquid phase of 85.9 pounds
ethanol, 5.9 pounds water, and 0.6 pounds polar lipids are recovered,
from which water and ethanol are removed by, e.g., evaporation, leaving a
polar lipid-rich product 425. Solvent recovered from the evaporation can
be recycled to the second extraction step 420.

[0157] A second solid phase from the second extraction step 420 is passed
to a third extraction step 430; this first solid phase includes 3.9
pounds of algae, 0.7 pounds of water, and 11 pounds of ethanol.
Seventy-found and a half pounds of ethanol and 3.5 pounds of water are
added to the second solid phase from the previous step. The third
extraction step 430 has a liquid phase of about 95.4% w/w ethanol. A
third liquid phase of 78.9 pounds ethanol, 3.9 pounds water, and 1.6
pounds neutral lipids are recovered, from which water and ethanol are
removed by, e.g., evaporation, leaving a neutral lipid-rich product 435.
Solvent recovered from the evaporation can be recycled to the second
extraction step 430 A solid phase of 2.3 pounds algae, 0.3 pounds water,
and 6.6 pounds ethanol remain.

[0158] As demonstrated in FIG. 12A, the resulting lipid profile with each
sequential ethanol extraction step was largely influenced by the moisture
content in the starting algae. Models of process 400 were run on three
different biomass collections, each having a different initial water
content. As the initial water content decreased, the maximum lipid
recovery step changed from the third extraction step to a fourth (not
shown). However, the overall lipid recovery from these three biomass
samples were quite similar, all above 95% of the total lipid content of
the algal biomass.

[0159] When algal mass with higher moisture content was used, the ethanol
concentration in the aqueous ethanol mixture was much lower, and
consequently the neutral lipid percentage in the crude extract was also
lower. It has been reported that dewatering an algae paste with 90% water
is a very energy intensive process. The methods described herein
unexpectedly can be used to successfully extract and fractionate an algal
mass containing mostly water. As overall lipid recovery was not
significantly influenced by starting from an algae paste containing 90%
water (10% algal solids), unlike conventional extraction methods, the
methods disclosed herein do not require the use of an energy intensive
drying step.

[0160] FIG. 12B shows an illustrative implementation 500 of one of the
extraction steps of process 400. An algae biomass and solvent mixture 505
is provided to an extraction vessel 510. After the algae is extracted (as
described elsewhere herein), the mixture is provided to a coarse
filtration system 515, such as a sintered metal tube filter, which
separates the mixture into a liquid phase and a solid phase. The solid
phase is passed to a downstream extraction step. The liquid phase is
passed to a solvent removal system 520, e.g., an evaporator, to reduce
the solvent (e.g., ethanol) content in the liquid phase. The liquid phase
remaining after solvent removal is, optionally, passed to a centrifuge
525. Any solids remaining in the solvent removal system are recycled or
discarded. Centrifuge 525 assists in separating the desired algal product
(e.g., proteins or lipids) from any remaining water and/or solids in the
liquid phase.

[0161] FIG. 14 shows an example of a process 600 by which an algal mass
can be processed to form or recover one or more algal products. In this
example, an algal biomass is extracted in a step-wise manner in a
front-end process 605 using the methods disclosed herein. The extraction
and separation steps are followed by an esterification process 610, a
hydrolysis process 615, a hydrotreating process 620, and/or a
distillation process 625 to further isolate components and products. The
components and products include algal lipids, algal proteins, glycerine,
carotenoids, nutraceuticals (e.g., long chain unsaturated oils and/or
esters), fuel esters (generally, the esters having chain lengths of C20
or shorter), fuels, fuel additives, naphtha, and/or liquid petroleum
substitutes. In preferred embodiments the fuel esters are C16 chain
lengths. In others, the fuel esters are C18 chain lengths. In still other
embodiments, fuel esters are a mixture of chain lengths, C20 or shorter.

[0162] The esterification process 610, hydrolysis process 615,
hydrotreating process 620, and distillation process 625 are optional and
can be used in various orders. The dashed arrows and dotted arrows
indicate some, but not all, of the options for when the hydrolysis,
hydrotreating, and/or distillation processes may be performed in the
processing of the lipid fractions. For example, in some embodiments of
the invention, after extraction and/or separation are carried out, the
neutral lipids fraction can be directly hydrotreated in order to make
fuel products and/or additives. Alternatively, in other embodiments, the
neutral lipid fraction can be passed to esterification process 610.

[0163] Esterification process 610 can include techniques known in the art,
such as acid/base catalysis, and can include transesterification.
Although base catalysis is not excluded for producing some products, acid
catalysis is preferred as those techniques avoid the soaps that are
formed during base catalysis, which can complicated downstream
processing. Enzymatic esterification techniques can also be used.
Esterification can process substantially pure lipid material (over 75%
lipid, as used herein). After esterification, glycerine byproduct can be
removed. The esterified lipids can then undergo molecular and/or
nonmolecular distillation (process 625) in order to separate esterified
lipids of different chain lengths as well as carotenoids present in the
lipid fraction. The esterified lipids can then be passed to hydrotreating
process 620 to generate jet fuel, biodiesel, and other fuel products. Any
hydrotreating process known in the art can be used; such a process adds
hydrogen to the lipid molecules and removes oxygen molecules. Exemplary
conditions for hydrotreating comprise reacting the triglycerides, fatty
acids, fatty acid esters with hydrogen under high pressure in the range
of 600 psi and temperature in the range of 600° F. Commonly used
catalysts are NiMo or CoMo.

[0164] Hydrotreating the fuel esters rather than the raw lipids has
several advantages. First, the esterification process 610 reduces the
levels of certain phosphorus and metals compounds present in algal oils.
These materials are poisons to catalysts typically used in hydrotreating
processes. Thus, esterification prior to hydrotreating prolongs the life
of the hydrotreating catalyst. Also, esterification reduces the molecular
weight of the compounds being hydrotreated, thereby improving the
performance of the hydrotreating process 620. Further still, it is
advantageous to retain the fuel esters from the distillation process 625
to be hydrotreated in a vaporous form, as doing so reduces the energy
needed for hydrotreating.

[0165] In some embodiments of the invention, the neutral algal lipids are
directly hydrotreated in order to convert the lipids into fuel products
and additives. While in other implementations, the neutral lipids are
esterified and separated into carotenoids, long chain unsaturated esters,
eicosapentaenoic acid (EPA) esters, and/or fuel esters via distillation
process 625. Distillation process 625 can include molecular distillation
as well as any of the distillation techniques known in the art. For
example, the distillates can be fractionated using a simple distillation
column to separate the lower chain fatty acids for refining. The long
chain unsaturated fatty acids remain as high boiling residue in the
column. In some embodiments, the remaining vapor can then be sent to the
hydrotreating process. Two of the advantages of the present invention are
that it yields pure feed as well as a vapor product, which favors the
energy intensive hydrotreating reaction, as described above.

[0166] In some embodiments of the invention, polar lipids (and,
optionally, neutral lipids) are hydrolyzed in hydrolysis process 615
before being passed to the esterification process. Doing so unbinds the
fatty acids of the algal lipids, and enables a greater amount of the
algal lipids to be formed into useful products.

[0167] FIG. 15 is a flowchart showing a process 700 for producing
nutraceutical products from neutral lipids. In one implementation of
process 700, neutral lipids are fed to an adsorption process 705 that
separates carotenoids from EPA-rich oil. The neutral lipids can be from
an algae source generated by any of the selective extraction techniques
disclosed herein. However, the neutral lipids can be from other sources,
such as plant sources.

[0168] Adsorption process 705 includes contacting the neutral lipids with
an adsorbent to adsorb the carotenoids, such as beta carotene and
xanthophylls. In one implementation, the adsorbent is Diaion HP20SS
(commercially available from ITOCHU Chemicals America, Inc.). The neutral
lipids can contact the adsorbent in a batch-type process, in which the
neutral lipid and adsorbent are held in a vessel for a selected amount of
time. After the contact time, the absorbent and liquid are separated
using techniques known in the art. In other implementations, the
adsorbent is held in an adsorbent bed, and the neutral lipids are passed
through the adsorbent bed. Upon passing through the adsorbent bed, the
carotenoids content of the neutral lipids is reduced, thereby producing
an oil rich in EPA.

[0169] The carotenoids can be recovered from the adsorbent material by
treating the adsorbent with an appropriate solvent, including, but not
limited to, alcohols such as ethanol, isopropyl alcohol, butanol, esters
such as ethyl acetate or butyl acetate, alkanes such as hexane, and
pentane.

[0170] FIG. 16 is a flowchart showing a process 800 for producing fuel
products 830 from neutral lipids 805. The neutral lipids can be from an
algae source generated by any of the selective extraction techniques
disclosed herein. However, the neutral lipids can be from other sources,
such as plant sources. The neutral lipids are treated in a degumming
process 810, in which the lipids are acid washed to reduce the levels of
metals and phospholipids in the neutral lipids. In some implementations,
a relatively dilute solution of phosphoric acid is added to the neutral
lipids, and the mixture is heated and agitated. The precipitated
phospholipids and metals are then separated from the remaining oil, for
example, by centrifuge.

[0171] The treated oil is then passed to bleaching process 815 to remove
chlorophylls and other color compounds. In some implementations,
bleaching process 815 includes contacting the oil with clay and or other
adsorbent material such as bleaching clay (i.e. bentonite or fuller's
earth), which reduce the levels of chlorophylls and other color compounds
in the oil. The treated oil then is passed to hydrotreating process 820,
which hydrogenates and deoxygenates the components of the oil to form
fuels products, for example, jet fuel mixtures, diesel fuel additive, and
propane. In addition, the hydrotreating process 820 also causes some
cracking and the creation of smaller chain compounds, such as LPG and
naptha. Any of the hydrotreating processes described herein can be used
for hydrotreating process 820.

[0172] The mixture of compounds created in the hydrotreating process 820
are passed to a distillation process 825 to separate them into various
fuel products 830. Distillation process 825 can include any of the
molecular and non-molecular distillation techniques described herein or
known in the art for separation of fuel compounds.

[0173] In some embodiments of the instant invention, proteins may be
selectively extracted from an algal biomass. Extraction of proteins using
the disclosed methods offers many advantages. In particular, algal cells
do not need to be lysed prior to extracting the desired proteins. This
simplifies and reduces costs of extraction. The methods of the instant
invention exploit the solubility profiles of different classes of
proteins in order to selectively extract and fractionate them from an
algal culture, biomass, paste, or cake.

[0174] For example, an algal biomass may be subjected to heating and
mixing to extract water and salt soluble proteins called albumins and
globulins. This mixture can then be subjected to a change in pH to
recover the alkali soluble proteins called the glutelins. This step can
then be followed by a solvent-based separation of the alcohol soluble
proteins called prolamins. The remaining biomass would be rich in
carbohydrates and lipids.

[0175] Proteins can be extracted from both saltwater and freshwater algal
cells, as shown in FIGS. 17 and 18. The presence of salt in the saltwater
algal culture or biomass affects the extraction of different classes of
protein, but the methods disclosed herein enable one to selectively
extract proteins from either fresh or saltwater algae.

[0176] In some embodiments, extraction of proteins from freshwater algal
cells is accomplished by the novel process shown in FIG. 17. Freshwater
algal cells or a freshwater algal biomass are heated and mixed. Mixing
can be accomplished by a variety of methods known in the art such as, but
not limited to, stirring, agitation, and rocking. This process generates
a first heated extraction mixture or slurry, comprised of a first
substantially liquid phase and a first substantially solid phase. The
solid and liquid phases are then separated. Separation can be
accomplished by a variety of methods known in the art including, but not
limited to, centrifugation, decantation, flotation, sedimentation, and
filtration. This first substantially liquid phase is enriched in albumin
proteins.

[0177] The first substantially solid phase is then mixed with salt water
and heated to generate a second heated extraction mixture or slurry,
comprised of a second substantially liquid phase and a second
substantially solid phase. The salt water may be natural seawater or may
be an aqueous salt solution. An example of such a solution would comprise
about typically 35 g/L comprising mainly of NaCl. The solid and liquid
phases are then separated. This second substantially liquid phase is
enriched in globulin proteins.

[0178] The second substantially solid phase is then mixed with water and
heated to generate a third heated extraction mixture or slurry, comprised
of a third substantially liquid phase and a third substantially solid
phase. The pH of this third extraction mixture or slurry is then raised
to about 9 or greater, enriching the third substantially liquid phase
with glutelin proteins. The solid and liquid phases are then separated,
the third substantially liquid phase being enriched in glutelin proteins.

[0179] The third substantially solid phase is then mixed with a solvent
set and heated to generate a fourth heated extraction mixture or slurry,
comprised of a fourth substantially liquid phase and a fourth
substantially solid phase. In one preferred embodiment, the solvent set
comprises ethanol. In other non-limiting embodiments, the solvent set
comprises one or more of the following solvents: methanol, isopropanol,
acetone, ethyl acetate, and acetonitrile. The solid and liquid phases are
then separated. This fourth substantially liquid phase is enriched in
prolamin proteins. The remaining fourth substantially solid phase may be
enriched in lipids, depending on the composition of the starting algal
biomass.

[0180] In some embodiments, extraction of proteins from saltwater algal
cells is accomplished by the novel process shown in FIG. 18. Saltwater
algal cells or a saltwater algal biomass are heated and mixed. Mixing can
be accomplished by a variety of methods known in the art such as, but not
limited to, stirring, agitation, and rocking. This process generates a
first heated extraction mixture or slurry, comprised of a first
substantially liquid phase and a first substantially solid phase. The
solid and liquid phases are then separated. Separation can be
accomplished by a variety of methods known in the art including, but not
limited to, centrifugation, decantation, flotation, sedimentation, and
filtration. This first substantially liquid phase is enriched in globulin
proteins.

[0181] The first substantially solid phase is then mixed with water and
heated to generate a second heated extraction mixture or slurry,
comprised of a second substantially liquid phase and a second
substantially solid phase. The solid and liquid phases are then
separated. This second substantially liquid phase is enriched in albumin
proteins.

[0182] The second substantially solid phase is then mixed with water and
heated to generate a third heated extraction mixture or slurry, comprised
of a third substantially liquid phase and a third substantially solid
phase. The pH of this third extraction mixture or slurry is then raised
to pH 9 or greater, enriching the third substantially liquid phase with
glutelin proteins. The solid and liquid phases are then separated, the
third substantially liquid phase being enriched in glutelin proteins.

[0183] The third substantially solid phase is then mixed with a solvent
set and heated to generate a fourth heated extraction mixture or slurry,
comprised of a fourth substantially liquid phase and a fourth
substantially solid phase. In one preferred embodiment, the solvent set
comprises ethanol. In other non-limiting embodiments, the solvent set
comprises one or more of the following solvents: methanol, isopropanol,
acetone, ethyl acetate, and acetonitrile. The solid and liquid phases are
then separated. This fourth substantially liquid phase is enriched in
prolamin proteins. The remaining fourth substantially solid phase may be
enriched in lipids, depending on the composition of the starting algal
biomass.

[0184] The disclosed methods also provide for the selective extraction of
different types of proteins, as shown in FIG. 17-20. Any of the steps of
the aforementioned extraction process can be performed separately from
the rest of the steps in order to selectively extract a single protein
product. Two examples of this appear in FIGS. 17 and 18, as the as
demonstrated by the dashed box around extraction step 1a.

[0185] In a non-limiting example, globulin proteins can be selectively
extracted from a freshwater algal biomass by mixing said biomass with
salt water and heating to generate a heated extraction mixture or slurry,
comprised of a substantially liquid phase and a substantially solid
phase. The solid and liquid phases can then be separated. The liquid
phase is enriched in globulin proteins. See FIG. 17, extraction step 1a.

[0186] In another non-limiting example, albumin proteins can be
selectively extracted from a saltwater algal biomass by mixing said
biomass with water and heating to generate a heated extraction mixture or
slurry, comprised of a substantially liquid phase and a substantially
solid phase. The solid and liquid phases can then be separated. The
liquid phase is enriched in globulin proteins. See FIG. 18, extraction
step 1a.

[0187] In a further non-limiting example, prolamin proteins can be
selectively extracted from either a freshwater or saltwater algal biomass
as shown in FIG. 19. The selective extraction is accomplished by mixing
the algal biomass with a solvent set and heating to generate a heated
extraction mixture or slurry, comprised of a substantially liquid phase
and a substantially solid phase. The solid and liquid phases can then be
separated. The liquid phase is enriched in prolamin proteins.

[0188] In yet another non-limiting example, a protein fraction can be
selectively extracted from either a freshwater or saltwater algal biomass
as shown in FIG. 20. The selective extraction is accomplished by mixing
the algal biomass with a solvent set to generate an extraction mixture or
slurry and effecting a pH change in the mixture. The mixture is comprised
of a substantially liquid phase and a substantially solid phase. The
solid and liquid phases can then be separated. The liquid phase is
enriched in proteins

[0189] Having been informed of these aspects of the invention, one of
skill in the art would be able to selectively extract a desired protein
from either a freshwater or saltwater algal biomass by either a single
step extraction process, or a multi-step extraction process. In light of
the instant disclosure, one of skill in the art would be able to
interchange the order of the above disclosed multi-step extraction
schemes, provided that the protein content of the algal mass and the
solubility properties of the proteins of interest are taken into account.
Other embodiments of the disclosed methods may incorporate a wash step
between each extraction step.

[0190] For any of the disclosed protein extraction methods, the extraction
mixture/slurry may be maintained at a heated temperature for a period of
time. In some embodiments, the extraction mixture is maintained at a
heated temperature for between about 20 minutes to about 90 minutes. In
some aspects, the extraction mixture is maintained at a heated
temperature for between about 20 minutes and about 60 minutes. In other
aspects, the extraction mixture is maintained at a heated temperature for
between about 45 minutes to about 90 minutes.

[0191] In some embodiments, the extraction mixture/slurry may be heated to
temperatures less than about 50° C. In some aspects, the albumin,
globulin, and glutelin proteins are extracted at temperatures of less
than about 50° C. In other embodiments the extraction
mixture/slurry is heated to a temperature close to the boiling point of
extraction mixture/slurry. In some aspects, the prolamin proteins are
extracted at temperatures close to the boiling point of the extraction
mixture/slurry. In other embodiments, the pressure is increased above
atmospheric pressure, up to and including, 50 psi, during the heating and
mixing steps to enhance extraction

Example 1

[0192] Green microalgae Scendesmus dimorphus (SD) were cultured in outdoor
panel photobioreactors. SD samples of varying lipid contents were
harvested. After removal of bulk water by centrifugation, the algal
samples were stored as 3-5 cm algae cakes at -80° C. until use. A
pre-calculated amount of wet algal biomass (15 grams dry algae weight
equivalent) and 90 mL of ethanol solvent was added into a three-neck
flask equipped with condenser, mechanical stirring and a thermocouple. In
one experiment, the mixture was refluxed for 10 min under microwave
irradiance. In a second, the mixture was refluxed for 1 h with electronic
heating. Afterwards, the mixture was cooled to room temperature and
separated into a diffusate and retentate by filtration.

[0193] The total lipids of algal samples were analyzed using a
chloroform-methanol-water system according to Bligh and Dyer's lipid
extraction method. This total lipid value was used as reference for the
lipid recovery calculation. Total lipids were further separated into
neutral lipids and polar lipids by standard column chromatography method
using 60-200 mesh silica gel (Merck Corp., Germany). Each lipid fraction
was transferred into a pre-weighed vial, initially evaporated at
30° C. using a rotary evaporator (Buchi, Switzerland) and then
dried under high vacuum. The dried retentates were placed under nitrogen
and then weighed. The fatty acid profile of each sample was quantified by
GC-MS after derivatization into fatty acid methyl esters using
heptadecanoic acid (C17:0) as the internal standard.

[0194] The results (data not shown) indicated that microwave assisted
extraction was best for removal of the polar lipids in the first
extraction step, and somewhat less effective for the separation of
neutral lipids. Electronic heating is more consistent in extraction
effectiveness. The final yield is comparable between microwave assisted
extraction and electronic heating assisted extraction, but, microwave
assisted extraction is significantly faster.

Example 2

Protein Extraction from Algal Biomass

[0195] (1) Acid Leaching: Algal biomass was soaked in water at pH 4.5 for
1 hour. The samples were then centrifuged at 3000 rpm for three minutes,
and the supernatant removed. The remaining solids were washed 3 times
with dilute acid (pH 4.5) and freeze dried.

[0196] (2) Alkaline extraction: Algal biomass was soaked in water at pH 11
for 1 hour. following the addition of pH-adjusted water. The samples were
then centrifuged at 3000 rpm for three minutes, and the supernatant
removed. The supernatant was neutralized with acid (pH 4.5) following the
centrifugation. The remaining solids were washed 3 times with dilute acid
(pH 4.5) and freeze dried.

[0197] The results of acid leaching and alkaline extraction are shown
below in Table 4.

[0198] Protein yield was calculated on a weight basis, comparing the
weight of the freeze dried solids to the weight of the algal biomass
prior to soaking in pH-adjusted water. Protein purity was determined by
the Official Method of the American Oil Chemists' Society (Ba-2a-38),
measuring the amount of nitrogen in the freeze dried solids of each
process. As proteins are an important product that adds to the value of
algal product extraction, this information allows for the use of
feedstocks with varying levels of protein in the systems and methods
disclosed herein.

Example 3

Extraction of Proteins from Saltwater Algal Biomass

[0199] The saltwater algal culture initially made up of about 1-10% w/w
solids in saltwater was heated to 50° C. and maintained at this
temperature for 1 hr. The resulting slurry was centrifuged to separate
the liquid phase from the solid phase. The liquid extract was determined
to be rich in globulin proteins (about 10% of the total proteins present
in the original algal biomass).

[0200] The solids were then suspended in fresh water and heated to about
50° C. and maintained for about 1 hour. The resulting slurry was
centrifuged again to separate the liquid from the solid phase. The liquid
phase was determined to be rich in albumin proteins (about 10% of the
total proteins present in the original algal biomass).

[0201] The solids were then suspended in ethanol to achieve a 70% w/w
mixture. This mixture was heated to about 75° C. and maintained at
that temperature for about 1 hour. The resulting slurry was centrifuged
to separate the liquid from the solid phase. The liquid phase was
determined to be rich in albumin proteins (about 30% of the total
proteins present in the original biomass).

[0202] The solids were then suspended in alkali solution (aqueous NaOH, pH
9) and heated to about 50° C. and maintained at that temperature
for about 1 hour. The resulting slurry was centrifuged to separate the
liquid from the solid phase. The liquid phase was determined to be rich
in glutelin proteins (about 50% of the total proteins present in the
original biomass).

Example 4

Step Fractionation and Extraction of Algal Biomass by Ethanol

[0203] One thousand pounds of Nannochloropsis biomass (cultured from
strain 202.0, obtained from Arizona State University, Laboratory for
Algae Research and Biotechnology, ATCC Deposit Number PTA-11048), was
harvested and dewatered until algae comprised about 35% w/w and then
finally frozen.

[0204] The extraction steps were performed in a 400 gallon jacketed kettle
with hinged lids. The lids were tied down with straps and sealed with
silicone. The system also contained a mixer with a 2 horsepower explosion
proof motor with a two blade shaft. The frozen algae material was emptied
into the tank and an equal weight of ethanol was pumped in using a
pneumatic drum pump. The material was stirred for 15 minutes and the
jacket heated with steam to obtain the desired temperature at each
extraction step. The desired temperature is near, meaning within
3° C. of the boiling point of the mixture, but not boiling. This
desired temperature is different at each extraction step as the boiling
point of the mixture changes as the proportion of ethanol is changed.
Upon reaching the desired temperature, the system was stirred
continuously held at the desired temperature for 60 minutes to ensure
that the contents of the kettle were uniformly heated.

[0205] The contents of the kettle were then pumped out of the extraction
vessel and into a Sharples decanter centrifuge, using a pneumatic Viking
vane pump at about 1 gallon per minute. The decanter centrifuge rotor
speed was set to about 6000 rpm. The solids were collected in an enclosed
plastic drum and consisted of about 50% w/w solids to liquids. These
solids were returned to the kettle, where the aforementioned extraction
steps were repeated. The liquid stream from the decanter was collected
into a feed tank was and then fed to the membrane filtration system. The
membrane used was a 0.375 ft2 SS membrane manufactured by Graver
Technologies. The operating conditions were 60° C.±5° C.
and with an average pressure gradient of 40 psi. The membrane system was
backwashed about every 15 minutes with compressed air to maintain the
flux. The permeate collected from the membrane system was free of any
particulate matter. The retentate was collected and recycled to the
decanter.

[0206] This extraction and fractionation is due to the change in polarity
of the solvent through the process in each extraction. In the extraction
shown in FIG. 13, the process began with about 1000 lbs. of wet algal
biomass containing about 65% pure water (35% w/w algal solids). This was
mixed with 860 lbs. of denatured ethanol (95% ethanol and 5% methanol),
resulting in a mixture containing about 55% aqueous ethanol. The solids
and liquids were separated using a decanter as described above. The wet
solid portion weighed 525 lbs. and was 40% dry mass. A total of 525 lbs.
of 95% the denatured ethanol was added to the solids, resulting in a
mixture made up of about 85% aqueous ethanol. The solids and liquids were
separated using a decanter as described above. The solid portion weighed
354.5 lbs. and was 40% dry mass. To this mass, another 700 lbs. of
denatured ethanol was added, resulting in a mixture of about 95% aqueous
ethanol. The solids and liquids were separated using a decanter as
described above. The resulting solids were about 40% dry mass. This
biomass requires 60% less energy to dry, calculated based on the latent
heat of water and ethanol.

[0207] In some experiments (data not shown) other types of denatured
ethanol were tried. Denatured ethanol containing 95% ethanol and 5%
isopropyl alcohol was used in an extraction, but was found not to be as
effective as 95% ethanol and 5% methanol. Use of 100% ethanol is a
preferred embodiment of the present invention, but is generally not
available due to cost constraints.

[0208] The permeate stream from the membrane system was evaporated using
an in-house fabricated batch still. The operating conditions were about
80° C. during the vacuum distillation. All of the ethanol in the
permeate was evaporated. These extraction steps were repeated three
times, resulting in four product pools, as shown in FIG. 13. This is
because with each extraction step, the polarity changed with the addition
of water to the mixture, allowing for the extraction of different
components with each step. Product 1 contained the algal proteins, and as
a result, retained excess water in the system that could not be vaporized
under the operating conditions. Product 2 contained the polar lipids.
Product 3 contained the neutral lipids. Finally, Product 4 was the
residual biomass, containing potential coproducts such as carotenoids.

Example 5

Dewatering and Extraction of Algal Biomass by Ethanol

[0209] Upon harvesting, algal biomass typically contains between about 0.1
to 0.5% (w/w) solids. This can be dewatered using any of the methods
known in the algae industry, including, but not limited to membrane
filtration, centrifugation, heating, sedimentation or flotation.
Flocculation can either assist in flotation or sedimentation. The typical
result of such methods is an algae slurry containing about 10% w/w
solids. To dewater further, another dewatering method may be used to
remove some of the remaining free water to get the concentration of
solids closer to 40% w/w. However, the cost of dewatering increases
exponentially after the first dewatering is carried out. An advantage of
the systems and methods disclosed herein is that the allow for the
extraction and fractionation of an algal mass that has undergone only one
round of dewatering.

[0210] An example of such a process might be that in the first extraction
round, following the protocol described in Example 3, 1000 lbs. of wet
biomass containing 90% pure water and is mixed with 1000 lbs. of
denatured ethanol (95% EtOH and 5% MeOH), resulting in a solvent mixture
of about 50% aqueous ethanol. The resulting biomass (350 lbs.) is 40%
dry. The solvent composition of these wet solids is 50% aqueous ethanol.
With another 350 lbs. of denatured ethanol, the composition of the
mixture would be about 81% aqueous ethanol. The resulting biomass (235
lbs.) is 40% dry. The solvent composition of these wet solids is 81%
aqueous ethanol. With another 470 lbs. of denatured ethanol, the
composition of the mixture would be about 95% aqueous ethanol. The
resulting solids would be 40% dry with about 95% ethanol. This wet
biomass requires 60% less energy to dry based on the latent heat of water
and ethanol. In this case, 100 lbs. of algae would have been extracted
using 1820 lbs. ethanol. When compared with Example 3, wherein the
starting material was 40% algal solids, 350 lbs. of the dry algae
equivalent was extracted with 2085 lbs. ethanol.